Staphylococcus aureus is the leading cause of a wide variety of deadly infections. The success of this pathogen is due to a number of factors, including its ability to grow as a biofilm, genetic resistance to multiple antibiotics, its diverseand highly-adaptable metabolism, and its impressive array of resistance mechanisms towards oxidative and nitrosative stress. Bacterial nitric oxide synthase (NOS) enzymes produce nitric oxide (NO) and have been implicated in resistance to exogenous oxidative stress and antibiotics in several bacteria, including S. aureus. Reactive oxygen species (ROS) are also produced naturally when bacteria undergo aerobic respiration, and we have observed increased endogenous ROS in a S. aureus nos mutant grown under aerobic respiration-promoting conditions. S. aureus NOS (saNOS) has previously been shown to protect against oxidative stress, antibiotics, and leukocyte-killing, and promotes survival in both murine abscess and sepsis models of infection. However, there still remain many central unanswered questions about this enzyme, including the potential function of NOS in bacterial physiology and endogenous stress resistance, as well as the mechanisms that govern nos gene expression and NOS enzyme activity. Our nos mutant also displays increased membrane potential, suggesting that saNOS regulates S. aureus metabolism by an as-yet unknown mechanism. Corresponding transcriptomic profiling from this growth condition also suggests that the nos mutant switches its gene expression from aerobic respiration to anaerobic respiration/fermentation pathways. These results collectively suggest that saNOS functions in S. aureus physiology to modulate respiration and endogenous oxidative stress. This hypothesis will be tested by dissecting the contribution of saNOS to metabolism (Aim 1) through the use of EPR to measure NO-cytochrome interactions, as well as assessing environmental (ROS, NO) and genetic (SrrAB) signals for their role in promoting the gene expression changes observed in the nos mutant. The response of nos gene expression to environmental cues that result in perturbations of the cell membrane, as well as to the two-component systems (TCS) LytSR, GraRS, and SrrAB will be assessed using a variety of genetic approaches (Aim 2). Finally, saNOS cellular reductase partner(s) will be identified by in vivo pull-down assays as well as screening of a transposon mutant library, followed by surface plasmon resonance (SPR) analysis of purified saNOS and potential interacting proteins to verify specific binding and association/dissociation kinetics (Ai 3). It is expected that this research will enlighten our understanding of the role of saNOS in regulating metabolism and endogenous oxidative stress resistance, mechanisms that contribute to the ability of S. aureus to adapt to different growth environments in vivo. In turn, this will ad in the development of novel treatment strategies against this formidable pathogen.